TLRs are type I transmembrane proteins composed of an extracellular domain of leucine-rich repeats and an intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain (Leulier and Lemaitre, Nat. Rev. Genet. 9:165-178 (2008)). Ten human and twelve mouse TLRs have been identified. Each TLR is able to recognize a particular molecular pattern. For instance, TLR2, 4, 5, 6 and 11 bind to bacterial outer membrane molecules such as lipopolysaccharide (LPS), peptidoglycan and lipoteic acid while TLR3, TLR7, TLR8 and TLR9 recognize bacterial, viral or even endogenous nucleic acids (Kawai and Akira, Semin. Immunol. 19:24-32 (2007)). Moreover, TLRs can be classified based on their cellular localization: TLR1, 2, 4, 5 and 6 are expressed on the cell surface while TLR3, 7, 8 and 9 are localized mostly, though not exclusively, in endosomal compartments (Kawai and Akira, Semin. Immunol. 19:24-32 (2007)).
When pathogens invade a host, innate immune cells such as macrophages, neutrophils, natural killer cells and dendritic cells recognize pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) through TLRs. TLR activation initiates intracellular signaling events that result in the expression of immune response genes including inflammatory and immune modulatory cytokines, chemokines, immune stimulatory receptors, which augments killing of pathogens and initiates the process of developing acquired immunity (Takeda and Akira, Int. Immunol. 17:1-14 (2005), Akira et al, Cell 124:783-801 (2006)). Inappropriate activation of some members of the TLR family, on the other hand, contribute to development of a variety of diseases including bacterial sepsis (TLR1, TLR2, TLR3, TLR4 and TLR9) (Wurfel et al, Am. J. Respir. Crit. Care Med. 178:710-720 (2008), Knuefermann et al, Circulation 110:3693-3698 (2004), Cavassani et al, J. Exp. Med. 205:2609-2621 (2008), Alves-Filho et al, Crit. Care Med. 34:461-470 (2006), Tsujimoto et al, J. Hepatol. 45:836-843 (2006)), non-infection systemic inflammatory response syndrome (TLR4) (Breslin et al, Shock 29:349-355 (2008)), multiple sclerosis (TLR3, TLR4 and TLR9) (Chen et al, Int. Immunopharmacol 7:1271-1285 (2007)), systemic lupus erythematosus (SLE) (TLR7 and TLR9) (Marshak-Rothstein and Rifkin, Annu. Rev. Immunol. 25:419-441 (2007)) and rheumatoid arthritis (TLR3, TLR4, TLR7, TLR8 and TLR9) (Choe et al, J. Exp. Med. 197:537-542 (2003), O'Neil, Nat. Clin. Pract. Rheumatol. 4:319-327 (2008)). Moreover, preclinical and clinical studies indicate that inhibition of TLR activity has therapeutic benefits for treating certain diseases. For example, diverse LPS-neutralizing agents and TLR4 antagonists have been evaluated to treat inflammatory diseases in animal and clinical studies (Leon et al, Pharm. Res. 25:1751-1761 (2008)). A TLR9 inhibitor, inhibitory CpG DNA (Plitas et al, J. Exp. Med. 205:1277-1283 (2008)), and an antagonistic anti-TLR3 antibody (Cavassani et al, J. Exp. Med. 205:2609-2621 (2008)) enhanced survival of a mouse with polymicrobial sepsis. Oligonucleotide-based TLR7 and TLR9 inhibitors prevented IFNα production from human plasmacytoid dendritic cells stimulated with serum from SLE patients (Barrat et al, J. Exp. Med. 202:1131-1139 (2005)). Unfortunately, the redundancy of the TLR family may limit the utility of inhibitors that target individual TLRs.
Upon stimulation, all TLRs recruit intracellular TIR-domain-containing adapters, such as TRIF and MyD88 (Kawai and Akira, Semin. Immunol. 19:24-32 (2007)). These adapter molecules mediate a downstream cascade of TLR-associated signaling. TRIF is recruited to TLR3 and TLR4, and appears to activate IRF3, MAPK, and NF-κB while MyD88 is associated with all TLRs, except TLR3, and phosphorylates IRAK, IRF5, IRF7, MAPK and NF-κB, which enhance the expression of type I IFN, inflammatory cytokine and IFN-inducible genes (Kawai and Akira, Semin. Immunol. 19:24-32 (2007)). Unlike other TLRs, endosomal TLRs, TLR3, 7, 8 and 9, all recognize microbial or host nucleic acids, as PAMPs or DAMPs, respectively. The redundancy and interconnectedness of the TLR signaling pathway suggests that it will be important to inhibit the activity of multiple TLRs simultaneously to effectively control inflammatory and autoimmune responses and to enhance the clinical efficacy of TLR antagonists as therapeutic agents.
It was discovered recently that certain cationic polymers are able to counteract the activity of a variety of oligonucleotide-based drugs (e.g., aptamers), irrespective of their nucleotide sequences (Oney et al, Control of Aptamer Activity by Universal Antidotes: An Approach to Safer Therapeutics, Nature Medicine (in press)). Moreover, immune stimulatory siRNA, a TLR7 agonist, condensed with a cyclodextrin-based polymer has been shown not to activate TLR7 (Hu-Lieskovan et al, Cancer Res. 65:8984-8992 (2005)). The present invention results, at least in part, from studies designed to determine whether agents that bind DNAs and RNAs in a sequence-independent manner (e.g., nucleic acid-binding cationic polymers) can neutralize endosomal TLR ligands and thereby inhibit activation of the corresponding TLRs.